Module 2.2 Technological Advancements Flashcards
Sanger Sequencing (Original Version)
Challenges
- Four sequencing reactions per sample
- Radioactive isotopes and toxic PAGE chemicals pose safety concerns
- Polyacrylamide gel handling is not easy (0.4mm thick and >40cm long)
- Manual interpretation of gel images is not scalable
Sanger Sequencing (2nd version)
Technological Advancements
- Fluorescent dyes replacing radioactive isotopes
- Capillary gel electrophoresis
- Computer processing of the data
- Improvement of the polymerase
- Molecular cloning techniques
- Polymerase chain reaction
Polymerase for DNA Sequencing
Characteristics (7)
- A DNA-dependent DNA polymerase.
- incorporate nucleotides rapidly
- High replicative fidelity
- High replicative processivity
- Function as a monomer (easier to purify)
- No or attenuated (reduced) 3’ exonuclease proofreading activity (avoid accidental 3’ nucleotide removal)
- Efficient incorporation of ddNTP
DNA Amplification Techniques
2 (so far)
- Molecular cloning
- Polymerase Chain Reaction (PCR)
Typical PCR requirements
6
- DNA template
- DNA polymerase (heat-resistant)
- DNA primers
- deoxynucleoside triphosphates, or dNTPs
- Buffer system
- Thermal cycler machine
Sanger sequencing
Fluorescent dye labeling
- fluorophores were selected to be able to distinguish between the dyes.
- emission wavelengths had to be well resolved from one another
- dyes had to be highly fluorescent to provide sufficient detection sensitivity
- can be either attached to the primer or directly attached to the deoxynucleotides.
fluorescent dye labeled primers
- sample to be sequenced needs to be split into four different reactions, with each reaction using one type of deoxynucleotide and one of the four fluorescent labeled primers
- reaction mixtures are then combined and an electrophoresis down a single polyacrylamide gel. The separated fluorescent bands of DNA can be detected and acquired directly by computer
fluorescent dye labeled ddNTPs
(dye terminators)
- fluorescent dyes are directly attached to the dideoxynucleotide
- Sequencing reaction takes place in a single reaction tube
- Run in a single polyacrylamide gel tube
- separated fluorescent bands of DNA can be detected and acquired directly by computer
capillary gel electrophoresis
procedure
-uses polyacrylamide gel field capillaries
- DNA migrates in capillary from cathode buffer (-) to anode buffer (+)
- separate as they migrate, laser located near output end of capillary excites fluorescent dye of DNA fragment and is read by detector.
- output of detector is sent to computer
- separated DNA fragments appeared as peaks with different migration times
capillary gel electrophoresis
benefits and drawbacks
Benefits:
- faster with better resolution and higher separation efficiency than a conventional polyacrylamide slab gel
- computer system detects and records dye fluorescence automatically and output data as fluorescent peak trace chromatograph
- System can directly sequence less than 1,000 nucletide long DNA fragments in a single reaction
Drawbacks:
- insufficient power of separation for resolving large DNA fragments >1KB that differ in length by only one nucleotide
fluorophore
any molecule or functional group which is capable of fluorescence
- all fluorochromes are fluorophores
fluorochrome
any fluorescent dye used to stain cells or tissue for microscopic examination
medium- to high-input capillary sequencing
- used capillary array electrophoresis instruments with 16 or 96 capillaries
- input ends of capillaries are arrayed spatially to accept samples directly from standard 96 well plates
- can sequence up to 384 DNA samples in a single batch.
-Batch rounds may occur up to 24 times a day - makes sequencing complex genomes, (eg. human genome) possible
DNA polymerase
processivity
the number of nucleotides incorporated during complex formation with a primary template DNA
- important to ensure that polymerase will be able to generate full DNA fragment and with the dideoxynucleotide
DNA polymerase
holoenzyme
a multi sub unit enzyme complex
-difficult to purify and reconstitute
-not useful in DNA sequencing chemistry
T7 Polymerase / DNA recognition complex
- from T7 bacteriophage
- requires a host factor, E. coli thioredoxin to function
- thermostable DNA polymerase isolated from a thermophilic bacterial microorganism
- T7 DNA polymerase does not distinguish ddNTP from dNTP and incorporates both nearly equal efficiencies.
- bands are significantly more uniform than in Sanger sequencing
T7 Polymerase
bacteriophage (aka phage)
a virus that infects and replicates within bacteria and archaea
Taq Polymerase
- more readily purified and modifiable for further improvement
-able to withstand high temperature - A double mutant form further improved dye terminator sequencing quality and accuracy.
- no intrinsic 3’ to 5’ exonuclease proofreading activity
- highly processive and active over a broad range of temperatures
molecular cloning requirements
- DNA of interest
- cloning vector to carry the DNA of interest
- host organism, usually a bacteria cell, that will grow and propagate the target DNA
- restriction enzymes
- DNA ligase
restriction endonuclease
A group of enzymes that recognize specific DNA sequences and cut double helix at specific sites defined by the local nucleotide sequence in a predictable manner
- can cleave a long double stranded DNA molecule into fragments of strictly defined sizes.
- have different sequence specificities
restriction enzymes
- naturally produced by bacteria as a defense mechanism against foreign DNA
- different species of bacteria make different restriction nucleases that degrade incoming viral DNA
- recognizes a specific palindromic sequence of 4-8 (often 6) nucleotides in DNA
usually cut the two strands of DNA at or near the recognition sites
restriction enzymes
sticky ends/ cohesive ends
-staggered cuts made by a restriction enzyme in the two strands of DNA which leave complementary overhangs at each end
- allow any two DNA fragments to be easily joined back together as long as the fragments were generated with the same restriction nuclease or with another nuclease that produces same cohesive ends.
restriction enzymes
blunt ends
- cuts made by restriction enzyme where cutting position is perfectly aligned without any overhang
- can be rejoined together even if made by different restriction enzymes
- efficiency of connecting two blunt ends usually lower than joining two cohesive ends
molecular cloning
DNA ligase
can join two blunt ends or two sticky ends together
recombinant DNA molecule
DNA molecule produced by splicing together two or more different fragments
plasmid
a small circular molecule of double stranded DNA derived from bacteria cells
pUC19 plasmid vector
- circular DNA that is 2686 base pairs in size
- contains a replication origin site (ori)
- can replicate independently in the host cells eg. E. coli cells.
- multicloning (MCS) site provides many restriction endonuclease recognition sites and can be cut easily with different commonly available restriction enzymes that allow insertion of a desired fragment
- carries ampicillin resistance (amp) gene and beta-galactosidase (lacZ) gene that produces blue colonies when lacZ substrate (IPTG) is present in culture media
molecular cloning
process
- cut the DNA of interest and the vector with the selected restriction enzyme
- covalently join DNA fragment to vector with DNA ligase and introduce the recombinant DNA into whole bacterial cells via transfection
-bacteria may take up plasmid with or without the DNA insert, or not at all - select for bacteria that have taken up the plasmid eg. colonies with ampicillin resistance from pUC19 plasmid
- select for bacteria that have taken up the DNA vector insert that disrupts lacZ gene expression and produces white colonies instead of blue colonies
- Colony with DNA of interest grown in large volume of culture media where cells double in numbers every 30 min and DNA targets can be replicated millions of times
- plasmid containing DNA of interest extracted and purified from lysed bacterial cells for DNA sequence
molecular cloning
transfection
the process of artificially introducing nucleic acids (DNA or RNA) into cells, utilizing means other than viral infection
Polymerase Chain Reaction
(PCR)
features
allows the DNA from a selected region of a genome to be amplified 1 billion fold in a cell free system, and effectively purify this DNA away from remainder of the genome
- after multiple cycles, the predominant PCR product will be the single species of DNA fragments between the two primers
- typically polymerizes 1,000 basis per minute, up to 30 cycles of reactions usually required for effective DNA amplification
- now used routinely to clone from genes of interest directly
PCR
Buffer system
solutions that provide suitable chemical environment for optimal activity and stability of the DNA polymerase
PCR
Thermal cycler machine
an automatic machine that expose reagents to repeated cycles of heating and cooling to permit different temperature-dependent reactions (eg. DNA melting and replication)
PCR
process
1 CYCLE:
- Denature: strands of the DNA double helix are exposed at high temperature and physically separated
- Annealing: DNA cooled in presence of large excess of the two primers that hybridize to the complementary regions in the two DNA strands.
- Extension/Elongation: primers synthesize a new DNA strand complementary to the DNA template strand by adding free nucleotides of the reaction mix to the 3’ end of the primers in the 5’ to 3’ direction.
